FAQ's for the Oris™ Cell Invasion Assay Kit.
How do I know if I have properly inserted the Oris™ Cell Seeding Stopper into each microplate well?
The tip of a properly inserted stopper will create a bullseye pattern at the bottom of the well. To view this bullseye pattern, turn the plate over after inserting the stoppers and tilt the plate at an angle. You will be able to see this bullseye pattern in the center of each well through the clear bottom surface. Stoppers that have not sealed well can be re-inserted until the tip is properly placed. Click here for a view of the bullseye pattern.
How reproducible is the Oris™ Cell Invasion Assay?
Results obtained using the Oris™ Cell Invasion Assay are highly reproducible when compared to other commercially available assays. Quality Control testing has indicated Coefficients of Variance (CV's) that range from 12%-24%.
What is the stability of the Oris™ Cell Invasion Assay Kit?
The Oris™ Cell Invasion Assay Kit is stable for a minimum of 6 months when the Oris™ Basement Membrane Extract (BME) is stored at -80°C and the remainder of the kit is stored at 4°C. If the Oris™ BME is stored at -20°C in a standard freezer, then the kit will only have a shelf life of 3 months. It is important to keep the Oris™ BME frozen until use since freeze-thaws will destroy the product integrity.
What cell lines can be used with the Oris™ Cell Invasion Assay Kit?
Any cell line that is adherent can be used with the Orisc Cell Invasion Assay Kit. The following are a few examples::
Are there any hints for adding my cells to the wells through the side ports of the Cell Seeding Stoppers?
Add or extract cells and media by placing the pipette tip along the wall of the well. Care should be taken not to disturb the Cell Seeding Stopper when introducing the pipette tip into the well. A gel loading tip may be useful.
Are there any hints for using the Oris™ Stopper Tool to remove the stoppers?
Yes, most importantly, do not use the stopper tool as a lever to pry the stoppers from the well at an angle, as doing so may cause displacement of the seeded cells. To remove the stoppers, first secure the 96-well plate by holding it firmly against the deck of your work space. Next, slide the tines of the stopper tool between the top of the stopper strip backbone, keeping the underside of the tool flush with the top surface of the plate. Finally, lift the tool vertically to gently remove the stopper.
In some of the wells, the contents were not uniformly distributed. Is there something that I can do to address this issue in the future?
Yes, lightly tap the plate on your work surface to evenly distribute well contents.
I typically aspirate media from the wells using a vacuum. Will this method work with the Oris™ Cell Invasion Assay?
No, due to the potential to dislodge the cell layer, we urge you not to aspirate media from the wells using a vacuum. Care should be taken throughout all steps of the assay not to disturb the cell layer.
How long can I let my invasion assay run?
The Oris™ BME overlay maintains the integrity of the gelled form in culture media for a maximum of 14 days at 37°C.
I diluted the BME solution with my inhibitor and the overlay never gelled. Why?
The BME overlay will not gel if the protein concentration solution is diluted below 9 mg/mL.
My BME overlay has bubbles in it after it gels? How do I remove them?
Once the BME has gelled, you will not be able to remove the bubbles. This is why care must be taken when pipetting to mix the BME solution before dispensing the overlay into each well.
How is the Oris™ Detection Mask attached to the 96-well plate?
Prior to adding any liquids to the wells of your 96-well plate, familiarize yourself with the attachment and removal of the Detection Mask. To do so, orient the chamfered corners of the mask with the chamfered corners of the 96-well plate, ensuring that the A1 corner of the mask is aligned with the A1 well of the plate. Then, align the holes in the mask attachment lugs with the pins on the bottom of the 96-well plate. Finally, gently press the mask into place ensuring that the mask is snapped flat against the bottom of the 96-well plate.
Can I attach the Detection Mask to any type of 96-well plate?
No, due to the engineering specifications of the Detection Mask, it will only attach to the plates supplied in the Oris™ Cell Migration Assay kit, the Oris™ Cell Invasion Assay kit, or the Oris™ Cell Migration Assembly kits.
Do I have to apply the Detection Mask at the beginning of an assay?
No, the mask may be applied at any point during the assay. For kinetic assays, it is often most convenient to apply the mask at the beginning of the assay before any liquids are placed in the well. For endpoint assays, using fixed and stained cells, it is often most convenient to apply the mask just before reading.
What if I forgot to attach the Detection Mask before I added my samples to the wells?
Once you have familiarized yourself with the technique of attaching and removing the migration mask, you may apply it after liquids have been placed in the wells.
Are the Oris™ Cell Seeding Stoppers reusable with new sterile plates?
No, the stoppers have been sterilized and Platypus can not guarantee the integrity of the stoppers after a second sterilization process since repeated autoclaving will cause damage to the stopper material.
Can the Oris™ Cell Seeding Stoppers be cleansed with ethanol or isoproponal?
No, alcohol cleansing may not effectively sterilize the stoppers due to the nature of the stopper material.
Can I use only a few of the wells and utilize the rest for another experiment(s) at a later date?
No. The assay must be performed with all 96-wells since the BME coating on the plate will degrade with frequent changes in temperature and humidity fluctuations.
Do I have to use a fluorescent stain and a fluorescence plate reader to obtain my results with the Oris™ Cell Invasion Assay Kit?
No, the Oris™ Cell Invasion Assay Kit has been designed to be used with any commercially available stain or labeling technique and the readout can be performed with a plate reader, digital imaging instrument or by microscopic examination (i.e., cell counting or image capture / analysis).
If I am using fluorometric readout of the results of the Oris™ Cell Invasion Assay, what type of a fluorescent dye would you recommend I use to label cells?
Fluorescent dyes derived from the fluorescein, rhodamine and cyanine families can all be used with these cell motility assays, so long as their emission wavelengths are within 490-700 nm. Note: the fluorescence stain used must uniformly stain the cells. The utilization of a fluorescence probe that is affected by experimental conditions will increase the variability of results and reduce the correlation between fluorescence signal and cell migration. Fluorescence probes that are affected by experimental conditions could be utilized as a counter-stain for the study of factors and processes that affect cell migration, invasion or 2-D closure.
Are there dyes that are incompatible with the Oris™ Cell Invasion Assay?
No, however, the fluorescence stain used must uniformly stain the cells. For transformed cells (siRNA, etc.,), you may see a population of cells in a well with varying degrees of staining depending on percent of transformation. Transformed cells can vary in their ability to uptake dye or express fluorescent fusion proteins.
Can cells be labeled with more than one dye?
Yes, as long as the emission wavelengths of both dyes fall between 490-700 nm (and the emission wavelength of the first dye does not overlap with the excitation wavelength of the second dye), and your detection equipment has appropriate filters to distinguish the dyes, multiplex detection is possible.
I have noticed that my background reading seems extremely high and upon further evaluation, the detection mask and 96-well plate seem to be contributing to the fluorescence. Is there something that I can do to address this issue?
Please take care not to contaminate the bottom of the 96-well plate and the detection mask with any substance that may autofluoresce. To remove dust and any other contaminants, wash the detection mask and the bottom of the 96-well plate with 70% ethanol and dry off.
Is the Oris™ Cell Invasion Assay compatible with top and bottom reading fluorescence plate readers?
The Oris™ Cell Invasion Assay is compatible with bottom probe plate readers. Examples of compatible machines include:
Will the entire Oris™ plate with lid fit in my plate reader?
Some plate readers have limited clearance and may not accept the plate routinely with the lid on. If you experience clearance problems, try removing the lid prior to measurement.
Oris™ is a trademark of Platypus Technologies, LLC
